Advertisement

Astrocytes pp 155-168 | Cite as

In Vitro Engulfment Assay to Measure Phagocytic Activity of Astrocytes Using Synaptosomes

  • Youkyeong Gloria Byun
  • Won-Suk ChungEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1938)

Abstract

Astrocytes eliminate unnecessary synapses, neural debris, and pathogenic proteins such as amyloid β plaque. Although the emerging evidences suggest that the phagocytic roles of astrocytes are critical in maintaining brain homeostasis during development as well as pathogenic conditions, the efficient assay for measuring phagocytic capacity and kinetics of astrocytes has been lacking. Here we present in vitro engulfment assay using purified astrocytes and synaptosomes. Based on imaging methods, either fluorescent or pH indicator-conjugated synaptosomes can be used in this assay.

Key words

Astrocytes Phagocytosis Engulfment Degradation pH indicator Synaptosomes Confocal microscope Live imaging 

Notes

Acknowledgments

The authors thank all members in Chung’s laboratory for helpful discussion. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (NRF-2016M3C7A1905391,NRF-2016R1C1B3006969, and NRF-2018R1A4A1020922) (W.-S. C).

References

  1. 1.
    Singh SK, Stogsdill JA, Pulimood NS, Dingsdale H, Kim YH, Pilaz LJ, Kim IH, Manhaes AC, Rodrigues WS Jr, Pamukcu A, Enustun E, Ertuz Z, Scheiffele P, Soderling SH, Silver DL, Ji RR, Medina AE, Eroglu C (2016) Astrocytes assemble Thalamocortical synapses by bridging NRX1alpha and NL1 via Hevin. Cell 164(1-2):183–196CrossRefGoogle Scholar
  2. 2.
    Allen NJ, Bennett ML, Foo LC, Wang GX, Chakraborty C, Smith SJ, Barres BA (2012) Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors. Nature 486(7403):410–414CrossRefGoogle Scholar
  3. 3.
    Xu J, Xiao N, Xia J (2010) Thrombospondin 1 accelerates synaptogenesis in hippocampal neurons through neuroligin 1. Nat Neurosci 13(1):22–24CrossRefGoogle Scholar
  4. 4.
    Chung W-S, Clarke LE, Wang GX, Stafford BK, Sher A, Chakraborty C, Joung J, Foo LC, Thompson A, Chen C, Smith SJ, Barres BA (2013) Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways. Nature 504(7480):394–400 http://www.nature.com/nature/journal/v504/n7480/abs/nature12776.html#supplementary-informationCrossRefGoogle Scholar
  5. 5.
    Iram T, Ramirez-Ortiz Z, Byrne MH, Coleman UA, Kingery ND, Means TK, Frenkel D, El Khoury J (2016) Megf10 is a receptor for C1Q that mediates clearance of apoptotic cells by astrocytes. J Neurosci 36(19):5185–5192CrossRefGoogle Scholar
  6. 6.
    Tasdemir-Yilmaz OE, Freeman MR (2014) Astrocytes engage unique molecular programs to engulf pruned neuronal debris from distinct subsets of neurons. Genes Dev 28(1):20–33CrossRefGoogle Scholar
  7. 7.
    Jones RS, Minogue AM, Connor TJ, Lynch MA (2013) Amyloid-beta-induced astrocytic phagocytosis is mediated by CD36, CD47 and RAGE. J Neuroimmune Pharmacol 8(1):301–311CrossRefGoogle Scholar
  8. 8.
    Ma Z, Stork T, Bergles DE, Freeman MR (2016) Neuromodulators signal through astrocytes to alter neural circuit activity and behaviour. Nature 539(7629):428–432CrossRefGoogle Scholar
  9. 9.
    Schafer DP, Lehrman EK, Kautzman AG, Koyama R, Mardinly AR, Yamasaki R, Ransohoff RM, Greenberg ME, Barres BA, Stevens B (2012) Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner. Neuron 74(4):691–705CrossRefGoogle Scholar
  10. 10.
    Penzes P, Cahill ME, Jones KA, VanLeeuwen JE, Woolfrey KM (2011) Dendritic spine pathology in neuropsychiatric disorders. Nat Neurosci 14(3):285–293CrossRefGoogle Scholar
  11. 11.
    Sekar A, Bialas AR, de Rivera H, Davis A, Hammond TR, Kamitaki N, Tooley K, Presumey J, Baum M, Van Doren V, Genovese G, Rose SA, Handsaker RE, Schizophrenia Working Group of the Psychiatric Genomics C, Daly MJ, Carroll MC, Stevens B, SA MC (2016) Schizophrenia risk from complex variation of complement component 4. Nature 530(7589):177–183CrossRefGoogle Scholar
  12. 12.
    Purice MD, Speese SD, Logan MA (2016) Delayed glial clearance of degenerating axons in aged Drosophila is due to reduced PI3K/Draper activity. Nat Commun 7:12871CrossRefGoogle Scholar
  13. 13.
    Ray A, Speese SD, Logan MA (2017) Glial Draper rescues Abeta toxicity in a drosophila model of Alzheimer's disease. J Neurosci 37(49):11881–11893CrossRefGoogle Scholar
  14. 14.
    Foo LC, Allen NJ, Bushong EA, Ventura PB, Chung WS, Zhou L, Cahoy JD, Daneman R, Zong H, Ellisman MH, Barres BA (2011) Development of a method for the purification and culture of rodent astrocytes. Neuron 71(5):799–811CrossRefGoogle Scholar
  15. 15.
    Brosius Lutz A, Chung WS, Sloan SA, Carson GA, Zhou L, Lovelett E, Posada S, Zuchero JB, Barres BA (2017) Schwann cells use TAM receptor-mediated phagocytosis in addition to autophagy to clear myelin in a mouse model of nerve injury. Proc Natl Acad Sci U S A 114(38):E8072–E8080CrossRefGoogle Scholar
  16. 16.
    Dunkley PR, Jarvie PE, Robinson PJ (2008) A rapid Percoll gradient procedure for preparation of synaptosomes. Nat Protoc 3(11):1718–1728CrossRefGoogle Scholar
  17. 17.
    Foo LC (2013) Purification of rat and mouse astrocytes by immunopanning. Cold Spring Harb Protoc 2013(5):421–432CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biological SciencesKorea Advanced Institute of Science and TechnologyDaejeonRepublic of Korea

Personalised recommendations